Synaptic integration of newly generated neurons in rat dissociated hippocampal cultures.

In the dentate gyrus of the hippocampus new neurons are born from precursor cells throughout development and into adulthood. These newborn neurons hold significant potential for self-repair of brain damage caused by neurodegenerative disease. However, the mechanism by which newborn neurons integrate into the brain is not understood due to a lack of knowledge of the molecular and functional characteristics of the synapses formed by newborn neurons. Here we report that dissociated hippocampal cultures continue to produce new granule cells in vitro that fire action potentials and become synaptically integrated into the existing network of mature hippocampal neurons. Quantification of the expression of synaptic proteins at newborn and mature granule cell synapses revealed synapse development onto newborn neurons occurs sequentially with initial synaptic contacts evident from 6 days after cell birth. These data also showed that the dendrites of newborn neurons have a high density of Piccolo and Bassoon puncta on them and therefore have a high potential to be integrated into the neuronal network through new synaptic connections. Electrophysiological recordings from newborn neurons reveal these synapses are functional within 10 days of cell birth. GABAergic input synapses were found to mature faster in newborn neurons than glutamatergic synapses where sequential recruitment of postsynaptic glutamate receptors occurred. Group I metabotropic glutamate receptors (mGluR1/5) were present at higher levels compared with ionotropic glutamate receptors (NMDA and AMPA receptors), suggesting that metabotropic and ionotropic receptors play differential roles at glutamatergic synapses in the integration and the maturation of newborn neurons. These data show that dissociated hippocampal cultures can provide a useful model system in which to study the integration of newborn neurons into existing neuronal circuits to increase our understanding of how the function of newborn neuron synapses could contribute to restoring damaged neuronal networks.

SAP97 directs NMDA receptor spine targeting and synaptic plasticity.

SAP97 is a multidomain scaffold protein implicated in the forward trafficking and synaptic localization of NMDA- and AMPA-type glutamate receptors. Alternative splicing of SAP97 transcripts gives rise to palmitoylated αSAP97 and L27-domain containing ßSAP97 isoforms that differentially regulate the subsynaptic localization of GluR1 subunits of AMPA receptors. Here, we examined whether SAP97 isoforms regulate the mechanisms underlying long-term potentiation (LTP) and depression (LTD) and find that both α- and ß-forms of SAP97 impair LTP but enhance LTD via independent isoform-specific mechanisms. Live imaging of α- and ßSAP97 revealed that the altered synaptic plasticity was not due to activity-dependent changes in SAP97 localization or exchange kinetics. However, by recording from pairs of synaptically coupled hippocampal neurons, we show that αSAP97 occludes LTP by enhancing the levels of postsynaptic AMPA receptors, while ßSAP97 blocks LTP by reducing the synaptic localization of NMDA receptors. Examination of the surface pools of AMPA and NMDA receptors indicates that αSAP97 selectively regulates the synaptic pool of AMPA receptors, whereas ßSAP97 regulates the extrasynaptic pools of both AMPA and NMDA receptors. Knockdown of ßSAP97 increases the synaptic localization of both AMPA and NMDA receptors, showing that endogenous ßSAP97 restricts glutamate receptor expression at excitatory synapses. This isoform-dependent differential regulation of synaptic versus extrasynaptic pools of glutamate receptors will determine how many receptors are available for the induction and the expression of synaptic plasticity. Our data support a model wherein SAP97 isoforms can regulate the ability of synapses to undergo plasticity by controlling the surface distribution of AMPA and NMDA receptors.